The serotonin derivative is a kind of N-(hydroxycinnamoyl)-amines. The N-(hydroxycinnamoyl)-amines are conjugates of hydroxycinnamic acid derivatives and aromatic amines. Feruloyltyramine, feruloyloctopamine, 4-coumaroyltyramine, feruloyl-3′-methoxyoctopamine, 4-coumaroyloctopamine, feruloylserotonin and coumaroylserotonin are the representative compounds of N-(hydroxycinnamoyl)-amines. The N-(hydroxycinnamoyl)-amines such as feruloyltyramine and coumaroyltyramine are involved in the strengthening of a plant cell wall through peroxidative polymerization with proteins in a cell membrane and lignin, a component of a cell wall (Schmidt et al., Planta, 205, 51–55, 1998), after being introduced in a plant cell wall. The amount of the compound synthesized in a plant rapidly increases by an invasion of pathogenic bacteria or wounding (Fritzermeier et al., Plant Physiol. 85, 34–41, 1987; Joos et al., Eur. J. Biochem. 204, 621–629, 1992).
One constituent of the N-(hydroxycinnamoyl)-amines, hydroxycinnamic acid derivatives is synthesized through phenylpropanoid pathway. In other words, hydroxycinnamoyl-CoA is synthesized from hydroxycinnamate by hydroxycinnamate:CoA ligase using ATP and CoA-SH. Feruloyl-CoA, cinnamoyl-CoA, 4-coumaroyl-CoA and sinapoyl-CoA are the representative ones.
The other constituent of the N-(hydroxycinnamoyl)-amines, aromatic amines is synthesized through shikimate pathway. That is, aromatic amines are produced by decarboxylation of an amino acid by decarboxylase. And tyramine derived from tyrosine; serotonin derived from tryptophan; dopamine; noradrenaline; and octopamine are the representative ones.
N-(hydroxycinnamoyl)-amines are produced by conjugation of hydroxycinnamic acid derivatives synthesized through phenylpropanoid pathway and aromatic amines synthesized through shikimate pathway using N-(hydroxycinnamoyl)transferase.
The tyramine N-(hydroxycinnamoyl)transferase (referred to as ‘THT’ hereinafter) is an enzyme producing N-(hydroxycinnamoyl)-amines using various hydroxycinnamic acid derivatives and aromatic amines as substrates. At first, THT was purified by chromatography after treating potato culture cells with an elicitor prepared from Phytophthora infestans (Hohifeld et al., Plant Physiol. 107, 545–552, 1995). The molecular weight of potato THT was 49,000, optimum pH was 6.5˜6.8 and optimum temperature was 55° C. Substrate specificity of the purified recombinant potato THT to hydroxycinnamic acid derivatives was investigated. As a result, substrate specificity to feruloyl-CoA was the best and substrate specificities to cinnamoyl-CoA, 4-courmaroyl-CoA and sinapoyl-CoA followed in that order. Substrate specificity of potato THT to amines was also investigated using feruloyl-CoA as a hydroxycinnamic acid derivative substrate. As a result, substrate specificity to tyramine was the greatest, and substrate specificities to octopamine, to dopamine and to noradrenaline were next in order. When coumaroyl-CoA was used as a substrate, substrate specificity of potato THT to tyramine was increased more than 10 times. Other amines such as tryptamine, agmatine, putricine, cadaverin, spermidine and spermine were not used as a substrate. And, THT worked as a competitive inhibitor of those substrates.
Tobacco cell culture solution was treated with pronase, an elicitor to induce THT enzyme activity. Then, tobacco THT was partially purified by chromatography (Negrel et al., Eur. J. Biochem. 247, 1127–1135, 1997). Based on the data of amino acid sequences of the purified enzyme, cDNA clone was obtained by the conventional method (Schmidt et al., J. Biol. Chem. 274, 4273–4280, 1999). 6 histidines were attached onto an amino-terminal of the above tobacco THT gene, which was expressed by E. coli expression vector pQ30. The expression of a target protein was induced by IPTG, and then the protein was purified by an affinity purification method, resulting in tobacco THT. The purified tobacco THT showed the best hydroxycinnamic acid derivative substrate specificity to cinnamoyl-CoA and feruloyl-CoA, and showed the best amine substrate specificity to octopamine when feruloyl-CoA was used as a donor.
THT gene was also cloned from a UV-irradiated hot pepper by differential screening technique (Back et al., Plant Cell Physiol. 42, 475–481, 2001). Substrate specificity of hydroxycinnamic acid derivatives of purified recombinant hot pepper THT was greatest to cinnamoyl-CoA and substrate specificities to sinapoyl-CoA, feruloyl-CoA and coumaroyl-CoA followed in that order. The maximum reaction velocity (Vmax) of cinnamoyl-CoA was 15 times higher than coumaroyl-CoA. Substrate specificity of hot pepper THT to amines was greatest to tyramine and next greatest to octopamine. The maximum reaction velocity (Vmax) of tyramine was 1.5 fold higher than octopamine. mRNA of hot pepper THT gene was expressed in every tissue and THT enzyme was over-expressed when a tissue was wounded.
As explained above, plant THT enzyme synthesizes various N-(hydroxycinnamoyl)-amines such as feruloyltyramine, feruloyloctopamine, 4-coumaroyltyramine, feruloyl-3′-methoxyoctopamine, 4-coumaroyloctopamine, by using hydroxycinnamic acid derivatives and amines as a substrate. But, there has been no such report so far that THT enzyme uses serotonin as its amine substrate.
In the meantime, serotonin derivatives such as feruloylserotonin and coumaroylsrotonin, a sort of N-(hydroxycinnamoyl)-amines, are known to be detected only in certain families of plant seeds by a very small amount. According to an earlier report, serotonin derivatives were separated from Carthamus tinctorius and had an anti-oxidant activity (Zhang et al., Chemical & Pharma. Bull. 44, 874–876, 1996). After then, an anticancer activity of serotonin derivatives was additionally reported (Kawashima et al., J. Interferron & Cytokine Res. 18, 423–428, 1998). And further, Korean patent No. 10-0354791 discloses a novel use of feruloylserotonin and coumaroylserotonin, an activity to promote bone-formation.
Although the usefulness of the serotonin derivatives was confirmed, a mechamism for biosynthesizing serotonin derivatives and a functional plant producing serotonin derivatives have not been reported yet. In addition, it has not been reported yet that THT enzyme can synthesize serotonin derivatives by using serotonin as an amine substrate.